Spaceflight-induced contractile and mitochondrial dysfunction in an automated heart-on-a-chip platform

Abstract

With current plans for manned missions to Mars and beyond, the need to better understand, prevent, and counteract the harmful effects of long-duration spaceflight on the body is becoming increasingly important. In this study, an automated heart-on-a-chip platform was flown to the International Space Station on a 1-mo mission during which contractile cardiac function was monitored in real-time. Upon return to Earth, engineered human heart tissues (EHTs) were further analyzed with ultrastructural imaging and RNA sequencing to investigate the impact of prolonged microgravity on cardiomyocyte function and health. Spaceflight EHTs exhibited significantly reduced twitch forces, increased incidences of arrhythmias, and increased signs of sarcomere disruption and mitochondrial damage. Transcriptomic analyses showed an up-regulation of genes and pathways associated with metabolic disorders, heart failure, oxidative stress, and inflammation, while genes related to contractility and calcium signaling showed significant down-regulation. Finally, in silico modeling revealed a potential link between oxidative stress and mitochondrial dysfunction that corresponded with RNA sequencing results. This represents an in vitro model to faithfully reproduce the adverse effects of spaceflight on three-dimensional (3D)-engineered heart tissue.

Keywords: heart-on-a-chip; microgravity; mitochondrial dysfunction; oxidative stress; spaceflight.

Fig. 1.

Schematic illustration of EHT fabrication and experimental timelines. hiPSCs were differentiated into cardiomyocytes which were then used to fabricate EHTs at 20 d postdifferentiation. EHTs were loaded into chambers and maintained for 3 wk before launch. Tissues remained on board the ISS for approximately 4 wk before being returned to Earth, at which time a subset of tissues were maintained for an additional week. RNA sequencing and TEM were performed at the conclusion of the experiment. Spaceflight and 1G ground control experiments were conducted with identical timelines.

Fig. 2.

Hybrid scaffolds and magnetic sensing allow for real-time automated force measurements of tissues with enhanced maturity. (A) Scaffolds used to generate EHTs are composed of dECM and rGO. (B) Tissue contraction was measured using a series of GMR sensors that convert magnetic moments to force. (C) Each EHT array was inserted into a sealed chamber that allowed for fluid and gas exchange. (D) Representative photograph of a chamber containing 6 tissues. (E) Representative photograph of the printed circuit board within the PHAB containing the GMRs. Using a reference sensor, magnetic noise could be reduced in the final signal. (F) Photograph of the experiment during a media exchange conducted by the crew while on board the ISS.

Fig. 3.

Impaired force production and increased arrhythmia in spaceflight EHTs. (A) Raw twitch force and (B) fold change in twitch force over time for ground controls and spaceflight samples. (C) The fit Gaussian distributions for Day 39 and Baseline twitch force timepoints indicating the Cohen’s d and difference in means, overlapping coefficient, and Cohen’s U3 for spaceflight (red) and ground controls (blue). Green lines in the Gaussian distributions indicate the means of each dataset. (D) Fold change in the change in beat interval and (E) raw change in beat interval over time for ground controls and spaceflight samples. There were initially 16 tissues for ground controls and 20 for spaceflight samples. Over time, the numbers decreased due to the scheduled preservation of tissues for imaging or RNA-seq at each time point, resulting in n = 4 at the end of the experiment for both the ground control and spaceflight samples. (F) The fit Gaussian distributions for Day 19 and Baseline change in beat interval timepoints indicating the Cohen’s d and difference in means, overlapping coefficient, and Cohen’s U3 for spaceflight (red) and ground controls (blue). Green lines indicate the means of each dataset. Data are mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = no significance as determined by a Welch’s t test) Time points chosen for further analyses (C and F) were chosen based on severity, as compensatory mechanisms identified later in the text may lead to recovery of interval irregularity at later timepoints.

Fig. 4.

Ultrastructural images and analysis reveals perturbations to sarcomere structure and mitochondrial function. (A) Representative TEM images of sarcomeres from hiPSC-CMs in Day 39 EHTs from ground and spaceflight experiments. (Scale bar, 500 nm.) (B) Analysis of sarcomere length and (C) Z-band width from for Day 39 spaceflight and control samples. The red dashed line indicates the median and the black solid lines indicate the upper and lower quartiles. n = 67 sarcomeres for ground controls (4 tissues, 15 to 17 replicates each) and n = 144 sarcomeres for spaceflight (4 tissues, 36 replicates each), P-values shown determined with Welch’s t test. (D) Representative TEM images of mitochondria (green) for Day 39 spaceflight and control samples. Red indicates circular cristae, and blue indicates compartmentalization in the cristae. (Scale bar, 500 nm.) (E) Analysis of mitochondrion area and (F) mitochondrion circularity. The red dashed line indicates the median; the black solid lines indicate the upper and lower quartiles. (G) Plot of mitochondrion circularity against area, with the Pearson Correlation Coefficient (r) and associated P-value shown. n = 58 mitochondria for ground controls (4 tissues, 15 to 17 replicates each) and n = 93 mitochondria for spaceflight (4 tissues, 36 replicates each), P-values shown in E and F determined with Welch’s t test. (H) Representative TEM image of intracellular lipid accumulations (yellow) found in Day 39 spaceflight samples. (Scale bar, 500 nm.)

Fig. 5.

RNA-seq analysis of spaceflight and ground control EHTs reveals significant expression level diversion of key genes and pathways. (A) Heatmap of the top differentially expressed genes with log2-fold change absolute values >4. (B) Canonical pathways projected to be of highest importance through IPA. Nodes colored blue were down-regulated or inhibited, nodes colored orange were up-regulated or activated, a solid line represents a direct interaction, and a dotted line represents an indirect interaction. Light blue lines indicate less confidence and dark blue lines indicate higher confidence. (C) Some of the top canonical pathways identified using IPA are visualized by activation z-scores (dot color) and –log(P-values) (dot size). Positive z-score indicates that the pathway is activated, and negative z-score indicates that the pathway is inhibited. (D) Normalized counts for genes associated with contractility, (E) ion channels, (F) mitochondrial function and metabolism (G) oxidative stress, and (H) inflammation. n = 11 tissues for spaceflight and n = 9 tissues for ground controls. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as determined by a Welch’s t test.)